大鼠涎腺放射损伤模型的建立及NBS1的表达变化

目的：研究大鼠涎腺放射损伤模型中NBS1基因的表达,初步探讨其在放射性涎腺上皮细胞损伤修复中的调控作用。方法：放射组40只大鼠以^60Coγ射线分次照射,每次3GY,隔天1次,共5次,累积剂量分别为3、6、9、12、15GY,对照组40只大鼠同期进行麻醉。照射后2-4h,收集大鼠腮腺,颌下腺。应用苏木素-伊红染色（HE）和透射电镜观察涎腺组织的显微和超微结构变化;应用逆转录聚合酶链技术（RT—PCR）检测腮腺、颌下腺NBS1mRNA的表达情况。结果：腮腺较颌下腺组织损伤严重;放射组和正常组比较,腮腺放射剂量9GY组起,颌下腺于12GY组起,NBS1mRNA表达水平减少（P〈0．05）。结论：大鼠涎腺放射损伤模型建立成功,NBS1可能参与涎腺放射损伤修复。     

α1-肾上腺素受体在颌下腺的表达及苯肾上腺素对唾液分泌调节的动物实验

目的研究α-肾上腺素受体亚型在家兔颌下腺的表达和分布，及其激动剂——苯肾上腺素促家兔颌下腺唾液分泌的相关机制。方法应用RT—PCR和Western blot检测家兔正常颌下腺α1-肾上腺素受体亚型mRNA及蛋白质的表达；免疫组化法检测颌下腺α1-肾上腺素受体亚型的分布及水通道蛋白5的表达；经颌下腺导管插管给予（1×10^-8）-（1×10^-6）moL／L的苯肾上腺素，观察家兔心率和血压的变化及促颌下腺唾液分泌的量效关系。结果家兔颌下腺有α1A-α1B和α1D-肾上腺素受体3种亚型的mRNA及蛋白质的表达，并广泛分布于导管和腺泡细胞的胞膜及胞质中。给予1×10^-7moL／L苯肾上腺素7d，促进颌下腺唾液分泌增加，而对心率、血压无明显影响；水通道蛋白5在腺泡及导管细胞的顶膜和侧膜的表达增加。结论家兔颌下腺存在α1A-、α1B-和α1D-肾上腺素受体3种亚型的mRNA和蛋白质的表达。经颌下腺导管给予低剂量苯肾上腺素促进唾液分泌是安全有效的；水通道蛋白5的表达增加可能与苯肾上腺素促唾液分泌的机制有关，该研究为临床治疗领下腺功能低下提供了初步依据。     

60Coγ射线照射大鼠涎腺组织后Mrell蛋白表达的初步研究

目的:检测Mre11基因及其蛋白在放疗后的大鼠腮腺和颌下腺组织中的表达变化。方法:选取近交系雄性Wistar大鼠60只,按单次照射0 Gy,3 Gy、6 Gy、9 Gy、12 Gy、15 Gy剂量分成6组,用60Coγ射线照射大鼠头颈部,2h后收集大鼠两侧腮腺和颌下腺组织。用透射电镜观察涎腺上皮细胞超微结构变化;用RT-PCR检测Mre11的基因表达情况;用免疫组化检测Mre11蛋白表达情况。结果:透射电镜显示:放射后大鼠腮腺腺泡细胞胞质和胞膜均出现不同程度的损坏,随着剂量的增加,这种损坏逐渐加剧,未见到再生、恢复的变化。颌下腺和导管变化不明显。RT-PCR检测Mre11mRNA表达无统计学差异。免疫组化检测Mre11蛋白表达有统计学差异。结论:治疗剂量的60Coγ照射对正常涎腺组织呈不可逆的损伤,损伤的程度具有剂量依赖性;大鼠涎腺细胞Mre11mRNA的表达无剂量依赖性;大鼠涎腺细胞Mre11蛋白的表达与辐射剂量以及组织类型有关,随剂量的增加先增强后减弱。     

Rad50在放射后大鼠涎腺中的表达

目的:检测Rad50在放射后大鼠涎腺组织中的表达水平。方法:选用60只Wistar大白鼠,随机分成6组,每组10只。分为对照组(未照射)、3 Gy组、6 Gy组、9 Gy组、12 Gy组、15 Gy组。放射组大白鼠用60Co一次性照射后2 h内处死,立即取出其腮腺、下颌下腺组织备用。用电镜观察放射后各组涎腺组织超微结构的变化,用反转录聚合酶链反应(RT-PCR)检测Rad50基因表达水平的变化,用免疫组织化学法观察其蛋白表达水平的变化。结果:各放射组涎腺组织中Rad50的表达均高于对照组(P<0.05),但不同放射剂量的各放射组之间Rad50的表达无明显差异(P>0.05)。透射电镜下可见大鼠腮腺、下颌下腺细胞超微结构随着放射剂量的增加,发生较明显改变。结论:放射可引起涎腺组织Rad50表达水平增高,但其表达水平并未随放射剂量的增加而增高,提示Rad50在涎腺放射损伤修复中的表达水平有限,这可能是涎腺放射敏感性机制之一。     

电离辐射对大鼠颌下腺钙离子激活型钾通道的影响

目的:通过研究电离辐射对大鼠颌下腺腺泡细胞膜上BK通道和IK通道开放的影响,探讨照射后唾液腺分泌功能降低的可能分子机制。方法:将大鼠随机分为对照组和照射组,对照射组大鼠的颌下腺区一次性15 Gy X线照射,对照组不照射。分别在照射后3 d、15 d和40 d取出大鼠颌下腺,胶原蛋白酶消化,从每只大鼠颌下腺消化液中挑选1个状态好的细胞,全细胞膜片钳技术研究大鼠颌下腺腺泡细胞膜上BK和IK1通道电流的变化。结果:1)在+80 mV时,相比对照组大鼠颌下腺腺泡细胞膜上BK通道电流密度,照射后3 d没有显著性差异,照射后15 d减少9.7%(P<0.05),照射后40 d减少13.8%(P<0.01)。2)在+80 mV时,相比对照组大鼠颌下腺腺泡细胞膜上IK1通道电流密度,照射后3d没有显著性差异,照射后15 d减少12.8%(P<0.05),照射后40 d减少17.1%(P<0.01)。结论:电离辐射所致的大鼠唾液腺分泌功能减退可能与辐射后大鼠颌下腺腺泡细胞膜上BK和IK1通道开放电流减少导致的K+外流减少有关。     

一次性18Gy放射对大鼠颌下腺损伤的实验研究

目的:观察一次性18 Gy放射对大鼠颌下腺组织学形态和唾液流率的改变。方法:40只大鼠随机分为实验组和对照组,每组20只。实验组一次性18 Gy局部照射大鼠颌下腺区域,对照组只麻醉不放射。8周后处死所有大鼠,处死前插管法提取大鼠颌下腺唾液,称取其质量,计算唾液流率,比较两组唾液流率的改变。处死后取颌下腺组织,经4%多聚甲醛固定,切片,HE染色,镜下观察颌下腺的组织学形态。结果:放射后实验组进食进水量下降、活动减少。实验组饮水频率高于对照组。对照组的唾液流率为(18.64±8.23)μL/min,实验组的唾液流率是对照组的57.42%,为(10.70±2.22)μL/min。HE染色显示,放射后实验组颌下腺细胞变性,间质血管充血,腺泡细胞内的空泡数量明显增多。结论:放射后大鼠颌下腺唾液流率明显降低,放射后8周,组织学形态出现显著变化。放疗对大鼠颌下腺组织学形态和唾液分泌功能的远期影响,尚需进一步观察和研究。     

SD大鼠下颌下腺主导管结扎损伤及再通后的组织学观察

目的:建立主导管结扎的SD大鼠下颌下腺组织损伤模型,观察主导管结扎及再通后腺体的组织学变化。方法:双重结扎SD大鼠下颌下腺主导管。部分腺体在6 d后摘取;其他腺体于6 d后松开结扎使再通,并于4、6、12、24 d后处死动物,取出腺体。通过HE及免疫组织化学染色观察其组织化学变化。结果:主导管结扎6 d后,腺体中腺泡消失,导管样结构增生,导管细胞表达CK19;在增生导管及周围见α6β1、LN阳性细胞。松开结扎再通的导管在6 d后有成熟的腺泡细胞出现,24 d后有大量成熟腺泡形成,Amylases、PAS(periodic acid-schiff)、AQP-5均在腺泡细胞中表达。结论:导管结扎损伤后腺体腺泡萎缩,导管上皮增生。松开结扎再通后腺泡细胞再生、功能恢复。     

下颌下腺细胞裂解液诱导骨髓间充质干细胞向涎腺细胞转分化的实验研究

目的：用下颌下腺细胞裂解液体外诱导骨髓间充质干细胞（bone marrow mesenchymal stem cells，BM—MSCs），观察BM—MSCs能否表达涎腺细胞的特异性分泌蛋白-α-淀粉酶。方法：分离并培养新生SD大鼠的下颌下腺细胞（submandubular gland cells，SMGCs），测定各代腺泡细胞分泌α-淀粉酶的量，取分泌量显著一代制成下颌下腺细胞裂解液；自新生SD大鼠股骨、胫骨中分离出BM—MSCs，培养至第3代时用含下颌下腺细胞裂解液的培养基培养1周，细胞免疫组织化学法鉴定诱导后的BM—MSCsa--淀粉酶的表达。结果：细胞免疫组化可检测到所培养的下颌下腺细胞中含α-淀粉酶，淀粉酶试剂盒测定结果显示，P2、P3代SMGCs分泌的α-淀粉酶较其它各代明显（P〈O．01）。经下颌下腺细胞裂解液诱导BM—MSCs后，可见下颌下腺腺泡样细胞，免疫组化显示其表达淀粉酶，而对照组未表达淀粉酶。结论：下颌下腺细胞裂解液能体外模拟下颌下腺细胞微环境诱导BM—MSCs转分化为具有分泌α-淀粉酶蛋白能力的下颌下腺腺泡样细胞，为解决涎腺组织工程中种子细胞的来源提供一条新途径。     

SSB1在腮腺放射性损伤修复过程中的表达变化

目的：研究正常大鼠腮腺及放射后大鼠腮腺组织单链DNA结合蛋白（ single?strand DNA?binding protein 1，SSB1）的表达及变化规律，探讨SSB1与涎腺放射损伤修复的关系。方法：取近交系成年Wistar大鼠40只，一次给予6 Gy剂量照射头颈部，于放射后每小时随机处死5只动物取腮腺组织，共形成8个实验组，另取5只大鼠作为对照组。应用免疫组织化学与RT?qPCR的方法检测SSB1在大鼠腮腺组织中表达情况。结果：对照组大鼠腮腺组织有一定量的内源性SSB1表达，不同腮腺组织之间SSB1含量不同，导管＞腺泡＞结缔组织；放射后，大鼠腮腺组织中SSB1的表达量随着时间变化呈先增加（P＜0?05）后减弱趋势，并在后期表达量降至接近正常水平。结论：辐射能诱导大鼠腮腺SSB1表达量增加，可能参与了大鼠腮腺放射性损伤修复过程。     

大鼠头颈部60Co照射后腮腺组织病理变化及临床意义

目的研究60Co照射对腮腺组织的影响及临床意义。方法模拟临床头颈放射治疗,用60Co对40只正常成年SD大鼠头颈部按剂量归一的方法进行照射。照射后分别于第5、10、20、30、60天取腮腺组织作常规病理切片观察。结果60Co照射后,随着时间的推移,大鼠腮腺腺泡细胞胞质肿胀、空泡变性和坏死明显加重,间质血管充血、肿胀及变性逐渐加剧,未见到再生、恢复的变化。结论治疗剂量的60Co照射对正常腮腺组织有损伤,主要作用于腺泡细胞和间质血管,损伤的程度具有时间依赖性,呈不可逆的损伤。     

Structural and functional injury in minipig salivary glands following fractionated exposure to 70 Gy of ionizing radiation: an animal model for human radiation-induced salivary gland injury

This study explored the feasibility of developing an animal model for radiation-induced salivary gland injury with a radiation protocol identical to current clinical practice. Three male Hanford minipigs were subjected to fractionated daily irradiation with a total dose of 70 Gy; structural and functional measures were compared with those of a control group of minipigs. We found that irradiated submandibular and parotid glands were one-third to one-half the gross size of control glands. Whereas no pathologic changes were noted in control glands, irradiated glands consistently demonstrated significant parenchymal loss with extensive acinar atrophy and interstitial fibrosis, enlarged nuclei in remaining acinar cells, and ductal dilatation and proliferation. Stimulated salivary flow was reduced by 81% in irradiated animals compared with preirradiation flow (P <.001); salivary flow in the control group increased by 30% during the same period (P <.001). The observed radiation-induced structural and functional salivary gland changes are comparable in every respect to those observed following irradiation of human salivary glands.     

电离辐射对大鼠颌下腺形态及功能的影响

目的：观察电离辐射对大鼠颌下腺形态及功能的影响。方法：一次性15Gy X射线局部照射大鼠唾液腺区，观察照射后30天内动物体重、颌下腺重量、毛果芸香碱的潜伏期（lag phase)、唾液流率、电解质、组织学等变化。结果：照射后大鼠体重及颌下腺重量减轻，唾液流率降低，Na^ 降低、K^ 升高，Ca^2 变化不显著、毛果芸香碱的潜伏期延长。早期主要是细胞形态结构的变化，晚期主要是唾液腺实质细胞萎缩，数量减少。结论：照射后大鼠颌吓腺发生了形态变化及不可逆性功能障碍。卷曲颗粒导管（CGT）细胞是靶细胞。     

Effects of phenylephrine on transplanted submandibular gland

Autotransplantation of the submandibular gland is a potential treatment for severe kerato-conjunctivitis sicca. However, one of the major barriers to this procedure is that secretions from the transplanted gland decrease shortly after the operation, which may lead to obstruction of Wharton's duct, or even to transplantation failure. Using a rabbit model, we investigated whether phenylephrine could improve the secretion from the transplanted gland. We found that phenylephrine treatment significantly reversed the decrease in salivary secretion after transplantation, enhanced the expressions of alpha1A-, alpha1B-, and alpha1D-adrenoceptor mRNA, and ameliorated atrophy of acinar cells. Furthermore, phenylephrine also induced translocation of aquaporin-5 from the cytoplasm to the apical membrane, and increased the levels of phospho-ERK1/2, ERK1/2, phospho-PKCzeta, and PKCzeta in the transplanted gland. These results indicate that phenylephrine treatment moderates structural injury and improves secretory function in the transplanted submandibular gland through promoting alpha1-adrenoceptor expression and post-receptor signal transduction.     

Underlying protective mechanism of α1-adrenoceptor activation against irradiation-induced damage in rat submandibular gland

Objectives

Damage to salivary gland after radiotherapy for head and neck malignant tumours can lead to irreversible oral complaints, which severely impair quality of life. The protective effect of α₁-adrenoceptor activation on the salivary glands after irradiation has previously been demonstrated. However, the exact mechanism remains unclear. In this study, we investigated the underlying cytoprotective mechanism of α₁-adrenoceptor activation in rat submandibular glands after irradiation.

Study design

Rats were locally irradiated using a linear accelerator in the head and neck region with a dose of 20 Gy. After irradiation, phenylephrine (5 mg/kg) was injected intraperitoneally for 7 successive days and the submandibular glands were then collected. The antiapoptotic effect of phenylephrine on the gland was examined by TUNEL, the proliferative cellular nuclei antigen (PCNA) was determined by immunohistochemistry, and the activation of c-Jun N-terminal kinase (JNK) was detected by Western blot.

Results

The irradiation only group showed severe atrophy, increased apoptosis, enhanced cell proliferation, and the phosphorylation of JNK was markedly increased by 26.89% (P < 0.05), compared to the control. The phenylephrine-treated group, however, showed remarkably alleviated atrophy, decreased apoptosis, and further increased cell proliferation, and the phosphorylation of JNK was markedly decreased by 36.00% (P < 0.05), compared to the irradiation only group.

Conclusions

The data showed that the underlying protective mechanism of α₁-adrenoceptor activation in irradiated gland might be related to improved cell proliferation, inhibited cell apoptosis, and depressed activation of JNK. It could be helpful in protecting salivary glands against irradiation damage.     

Effect of x-ray irradiation on lipid peroxide levels in the rat submandibular gland

We examined the effect of local x-ray irradiation on the changes in the lipid peroxide level in submandibular gland, liver, and blood plasma. Rats five weeks of age received a single low dose of 3 Gy x-ray irradiation to their neck regions. The lipid peroxide level in the submandibular gland was significantly enhanced at seven days after irradiation, as was the level in the blood plasma at two hours, seven and 14 days after irradiation. The lipid peroxide level in the liver decreased, as compared with levels in the controls. There was a slight tendency for acinar cells of the submandibular gland to show pyknosis and anomalous nuclei within three days after irradiation. These results suggest that radiation injury results in an elevation of the lipid peroxides in the submandibular gland, and an increased level in the blood.